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- PDB-6jcw: Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductois... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6jcw | |||||||||
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Title | Cryo-EM Structure of Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) with Mg2+ at pH8.5 | |||||||||
![]() | ketol-acid reductoisomerase | |||||||||
![]() | ISOMERASE / Bi-specific / Thermostable / Reductoisomerase / Magnesium-dependent / Dodecamer / Knotted protein | |||||||||
Function / homology | ![]() ketol-acid reductoisomerase (NADP+) / ketol-acid reductoisomerase activity / valine biosynthetic process / isoleucine biosynthetic process Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å | |||||||||
![]() | Chen, C.Y. / Chang, Y.C. / Lin, K.F. / Huang, C.H. / Lin, B.L. / Ko, T.P. / Hsieh, D.L. / Tsai, M.D. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase. Authors: Chin-Yu Chen / Yuan-Chih Chang / Bo-Lin Lin / Kuan-Fu Lin / Chun-Hsiang Huang / Dong-Lin Hsieh / Tzu-Ping Ko / Ming-Daw Tsai / ![]() Abstract: While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 ...While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg. The results showed that the distances of the two catalytic Mg ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology. | |||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 653.2 KB | Display | ![]() |
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PDB format | ![]() | 550.9 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 9799MC ![]() 9798C ![]() 9800C ![]() 9801C ![]() 6jcvC ![]() 6jczC ![]() 6jd1C ![]() 6jd2C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 37229.855 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: ilvC2, ilvC-2, SSO1322 / Production host: ![]() ![]() #2: Chemical | ChemComp-MG / #3: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Sulfolobus solfataricus ketol-acid reductoisomerase (Sso-KARI) Dodecamer in complex with Mg2+ at pH8.5 Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.446 MDa / Experimental value: YES | ||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 8.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 0.58 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k) |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43916 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 117.95 / Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6JCV Pdb chain-ID: A / Accession code: 6JCV / Source name: PDB / Type: experimental model |